A METHOD FOR RECOVERING
VIRUSES FROM SLUDGES
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EPA-600/J-81-545
Journal of Virological Methods, 3 (1981) 283-291 283
Elsevier/North-Holland Biomedical Press
A METHOD FOR RECOVERING VIRUSES FROM SLUDGES
DONALD HERMAN*, GERALD BERG and ROBERT S. SAFFERMAN
Virology Section, Biological Methods Branch, Environmental Monitoring and Support Laboratory,
Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH 45268,
U.SA.
(Accepted 17 August 1981)
Primary, activated, and anaerobic mesophilically digested sludges were salted with MgCl2 (divalent
cations) or A1C13 (trivalent cations) and acidified to bind indigenous unadsorbed virions to the sludge
solids; the sludges were centrifuged, and the adsorbed virions were eluted from the solids with buf-
fered 10% beef extract. The elution yields with this procedure were superior to those obtained from
sludges that had been salted or acidified only. Homogenization of sludges prior to other treatment did
not increase the numbers of virions recovered.
enterovirus virus recovery wastewater sludge testing procedure
INTRODUCTION
Virions adsorbed from water onto cellulose nitrate or fiberglass filters may be eluted
more readily with buffered 3% beef extract than by many other methods. Elution of
virions from such filters is achieved at least as efficiently with buffered 3% beef extract
as with buffered beef extract concentrations up to 15% (Berg et al., 1971). Virions ad-
sorbed to river water solids may also be eluted with buffered beef extract, but virions are
more readily eluted from such solids with buffered 10% beef extract than with buffered
3% beef extract (Berg and Dahling, 1980).
This paper describes a beef extract elution method for recovering virions from pri-
mary, activated, and anaerobic mesophilically digested sludges.
MATERIALS AND METHODS
Buffered beef extract (BE)
An appropriate quantity of powdered beef extract (Lab-Lemco), 1.34gofNa2HP04 -7
* Present address: Microbiological Treatment Branch, Municipal Environmental Research Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH 45268.
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H20, and 0.12 g of citric acid were mixed with 100 ml of distilled water and autoclaved
at 121°C for 15 min. The pH of the buffered beef extract (BE) was 7.0 ± 0.1. The eluting
capacity of each new lot of BE was compared to that of lots of known eluting capacity to
determine that the capability of each new extract to elute at least several enteroviruses
(poliovirus 1, coxsackievirus A9, and echovirus 7) was maximal (Berg et al., 1971).
Cell cultures
Viruses recovered from sludges were assayed in a continuous line of African green
monkey kidney cells designated BGM (Dahling et al., 1974). The line was propagated in a
medium of equal parts Eagle's minimal essential medium (MEM) in Hanks' balanced salt
solution (HBSS) with L-glutamine and non-essential amino acids (Grand Island Biological
Co.) and L-15 medium (Leibovitz) with L-glutamine (KC Biological), to which were
added calf serum (final concentration = 10%) and NaHC03 (final concentration = 0.075%)
(Dahling and Safferman, 1979). The cells were grown in 6 oz prescription bottles. Con-
fluent monolayers were washed once on the day of the test with Earle's balanced salt
solution and then inoculated.
Assays for virions
Virions were assayed by the plaque technique (Dahling et al., 1974). The cells used in
this study were in passages 126—150. Drained cultures were inoculated with 1 ml of
eluate, and the cultures were incubated at room temperature for 2 h. The inoculated
cultures were then overlayed with a medium each 100 ml of which contained 41.5 ml
2 X MEM in HBSS with L-glutamine and non-essential amino acids (Grand Island Biological
Co.), 2 ml heat-inactivated fetal calf serum, NaHCO3 (final concentration = 0.225%),
MgCl2 (final concentration = 0.01%), neutral red (final concentration = 0.0015%), 1 ml
sterile whole milk (Real Fresh Milk, Inc.) added shortly before overlay, 10,000 units of
penicillin, lOmg of streptomycin, 1.25 mg of tetracycline, 0.1 mg of amphotericin B,and
50 ml of a 3.0% solution of autoclaved Bacto Agar (Difco). The liquid part of the medium
was filtered through a 0.22 /mi Millipore HA membrane filter and warmed to 37°C before
the agar, cooled to 43°C, was added. Plaques were counted periodically and marked
permanently when recorded.
Sludges
Primary, activated, and anaerobic mesophilically digested sludges were obtained from
six sewage treatment plants in southwest Ohio. Sludges were transported to the labo-
ratory within 2 h of collection, hand-shaken until well-mixed, and appropriate volumes
were dispensed into beakers. Sludges were stored overnight at 4°C.
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pH adjustment of sludges
In some experiments, the pH of the sludge was adjusted to 3.0-3.5. In these instances,
the pH was checked for drift 15 and 30 min after adjustment and readjusted to 3.0-3.5 if
necessary. There was usually little pH drift between 15 and 30 min after the initial pH ad-
justment.
Suspended solids
Suspended solids were determined by a standard method (American Public Health
Assoc., 1975).
Membrane stacks
Membrane stacks consisted of 3.0, 1.2, 0.6 and 0.45 /Ltm Millipore MF membrane fil-
ters, in that order, stacked together in one filter holder. The filter stacks were used to
remove bacteria and fungi from BE eluates.
RESULTS
Recovering virions from sludges
Recovery of virions from primary and activated sludges. To determine whether buffered
10% BE would elute viruses from sludges as it did from river water solids (Berg and Dah-
ling, 1980), sufficient buffered 50% BE was mixed with 100 ml volumes of primary and
activated sludges to yield final BE concentrations of 10% in the sludges. The treated slud-
ges were centrifuged, and the virions within the supernatants were assayed. Virions, often
numbering in hundreds, were recovered from both primary and activated sludges.
Recovery of virions from blended primary and activated sludges. Sludge was blended in a
Waring blender (Model PBS) at maximum speed for 1 or 4 min. 25 ml of buffered 50%
BE were then mixed with 100 ml samples of both blended and non-blended sludge. The
final concentration of BE in the samples was 10%. The mixtures were stirred on mag-
netic stirrers for 30 min and then centrifuged at 10,000 X g for 30 min. The solids were
discarded, the supernatants were filtered through a membrane stack, and the virions with-
in the supernatants were assayed.
In four comparative tests, greater numbers of virions were recovered from unblended
primary sludges than from blended primary sludges (Table 1). In a similar series with ac-
tivated sludges, three of five unblended sludges yielded greater numbers of virions than
blended samples did. Thus, blending for 1 or 4 min did not increase the numbers of
virions recovered from sludges and may have even decreased them somewhat.
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TABLE 1
Recovery of indigenous virions from blended and unblended sludges
Sewage
treatment
plant
Lebanon
Muddy Creek
Sycamore
Taft
Lebanon
Muddy Creek
Taft
Sycamore
Type
of
sludge
Primary
Activated
Blending
time
(min)
1
1
4
4
1
1
4
4
4
Virions recovered
Blended
(p.f.u./lOOml)
562
200
27
512
112
187
205
408
15
Unblended
(p.f.u./lOO ml)
608
212
70
569
108
229
240
387
25
Recovering virions concentrated onto sludge solids
Since solids usually constitute less than 6% of the volume of sludges, and since many
of the virions in sludges are adsorbed to these solids even before the sludges are formed,
it seemed that the best approach to maximizing the recovery of virions from sludges
should center on adsorbing the virions suspended in the liquid part of the sludges onto
the solids, concentrating the solids, and then eluting the adsorbed virions from the solids.
Since cations, especially in acid solution, often increase the adsorption of viruses to
solids, salts were added to each sludge, the pH of the sludge was adjusted to 3.0—3.5
(Berg et al., 1971; Wallis and Melnick, 1967), the sludge solids were separated by cen-
trifugation, and adsorbed virions were eluted from the solids.
Recovery of virions from acidified primary and activated sludges and from MgCl2-
salted, acidified primary and activated sludges
MgCl2 • 6H2O was mixed into one of two paired 100 ml samples of primary sludge
(final MgCl2 molarity = 0.05). The pH of each sample was then adjusted to 3.0—3.5 with
5 N HC1. Paired 100 ml samples of activated sludge were treated similarly. The paired
samples were mixed with magnetic stirrers for 30 min and centrifuged at 2500 X g for 15
min. Supernatants were discarded, the solids of each sample were suspended in 100 ml
quantities of buffered 10% BE, and the suspensions were mixed on magnetic stirrers for
30 min and centrifuged at 10,000 X g for 30 min. The supernatants were filtered through
membrane stacks and the virions within the supernatants were assayed.
In six of seven tests, the presence of MgCl2 increased the yields of virions from acidified
primary sludges above those obtained from acidified sludges to which MgCl2 had not
been added (Table 2). Similar results were obtained with three of five paired samples of
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TABLE 2
Effect of divalent cations (Mg2+ as MgCl2) on recovery of indigenous virions from sludges acidified
to pH 3.0-3.5
Sewage Type
treatment of
plant sludge
Taft Primary
Sycamore
Mill Creek
Lebanon
Muddy Creek
Taft Activated
Sycamore
Lebanon
Muddy Creek
Mill Creek
Total
suspended
solids
(g/ 100 ml)
1.98
0.27
3.95
3.07
1.13
1.16
1.00
0.63
0.50
0.56
1.09
0.99
Virions recovered
Acidified sludge
without MgCl2
(p.f.u./lOOml)
468
188
80
220
427
713
360
380
27
313
247
87
Acidified sludge
with MgCl2
(p.f.u./lOOm!)
558
152
107
312
450
910
427
292
27
353
280
113
activated sludges. With one of these paired samples, there was no difference. The numbers
of virions recovered from acidified primary and activated sludges (to which MgCl2 had
not been added) with the technique described were almost always considerably greater
than those recovered from parallel samples by the 50% BE extraction procedure described
above (data not shown).
Recovery of virions from MgO2-salted and from MgCl^-salted, acidified anaerobic
mesophilically digested sludges
MgCl2 • 6H20 was mixed into two 100 ml samples of anaerobic mesophilically di-
gested sludge (final MgCl2 molarity = 0.05). One of the samples was subsequently acidi-
fied with 5 N HC1 to pH 3.0-3.5. Each sludge sample was mixed for 30 min and cen-
trifuged at 2500 X g for 15 min. The supernatant was discarded, the solids were sus-
pended in 100 ml of buffered 10% BE, and the suspended solids were mixed for 30 min
and centrifuged at 1C,000 X g for 30 min. The supernatant was filtered through a mem-
brane stack, and the virions within the supernatant were assayed.
With four of the five paired samples tested, greater yields of virions were obtained
from MgCl2-salted anaerobic mesophilically digested sludges subsequently acidified to pH
3.0-3.5 than from salted anaerobic mesophilically digested sludges that had not been
acidified (Table 3).
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TABLE 3
Effect of acidification to pH 3.0-3.5 on recovery of indigenous virions from anaerobic mesophilic-
ally digested sludges to which divalent cations (Mg2+ as MgCl,,) had been added
Sewage
treatment
plant
Mill Creek
Lebanon
Sycamore
Dayton
Total
suspended
solids
(g/ 100 ml)
6.67
5.23
2.30
0.17
6.03
Virions recovered
MgCl2 added,
sludge not acidified
(p.f.u./lOOml)
63
132
303
30
72
MgCl2 added
sludge acidified
(p.f.u./lOO ml)
87
345
217
80
77
Recovery of virions from AlCl3-salted and from AlCl3-salted, acidified anaerobic
mesophilically digested sludges
A1C13 • 6H2O was mixed into two 100 ml samples of anaerobic mesophilically digested
sludge (final A1C13 molarity = 0.0005). One of the samples was subsequently acidified
with 5 N HC1 to pH 3.0-3.5. Each sludge sample was mixed for 30 min and centrifuged
at 2500 X g for 15 min. The supernatant was discarded, the solids were suspended in 100
ml of buffered 10% BE, and the suspended solids were mixed for 30 min and centrifuged
at 10,000 X g for 30 min. The supernatant was filtered through a membrane stack, and
the virions within the supernatant were assayed.
With all five paired samples tested, greater yields of virions were obtained from A1C13-
salted anaerobic mesophilically digested sludges subsequently acidified to pH 3.0—3.5
than from salted anaerobic mesophilically digested sludges that had not been acidified
(Table 4).
Relative effectiveness of MgCl2 and AIC13 for recovering indigenous viruses from
acidified primary, activated, and anaerobic mesophilically digested sludges
MgCl2 • 6H2O and A1C13 • 6H2O were mixed into separate 100ml samples of sludges
(final MgCl2 molarity = 0.05, final A1C13 molarity = 0.0005). The pH of each sludge
sample was adjusted to 3.0-3.5 with 5 N HC1, and each sample was mixed for 30 min
and centrifuged at 2500 X g for 15 min. The supernatant of each was discarded, the
solids were suspended in 100 ml of buffered 10% BE, and the suspended solids were
mixed for 30 min and centrifuged at 10,000 X g for 30 min. Each supernatant was fil-
tered through a membrane stack, and the virions within the supernatant were assayed.
With three of four primary sludges, MgCl2 produced greater recoveries of virions than
A1C13 did. With two of three activated sludges, A1C13 produced greater recoveries of
virions than MgCl2 did. With three of five anaerobic mesophilically digested sludges,
MgCl2 produced greater yields of virions than A1C13 did (Table 5). Thus, MgCl2 (final
molarity = 0.05) and A1C13 (final molarity = 0.0005) produced about equal results.
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TABLE 4
Effect of acidification to pH 3.0-3.5 on recovery of indigenous virions from anaerobic mesophilic-
ally digested sludges to which trivalent cations (A13+ as AlCl.) had been added
Sewage
treatment
plant
Mill Creek
Lebanon
Sycamore
Dayton
Total
suspended
solids
(g/ 100 ml)
6.67
5.23
2.30
0.17
6.03
Virions recovered
A1C13 added,
sludge not acidified
(p.f.u./100ml)
50a
151
216
27
49
A1C13
sludge
(p.f.u.
63a
377
336
76
69
added,
acidified
/100ml)
a A1C13 was added as a solid (1.2 g A1C13
0.05 M.
6H2O/100 ml sludge). The final A1C13 concentration was
TABLE 5
Relative effectiveness of Mg2+ (as MgCl2) and A13+ (as A1C13) for recovering indigenous virions from
acidified (pH 3.0-3.5) sludges
Sludge
Primary
Activated
Anaerobic
mesophilically
digested13
Sewage
treatment
plant
Muddy Creek
Sycamore
Muddy Creek
Sycamore
Mill Creek
Lebanon
Sycamore
Dayton
Total
suspended
solids
(g/ 100 ml)
4.01
6.30
6.81
2.70
1.32
0.61
0.92
5.23
6.67
2.30
0.17
6.03
Virions recovered
MgCI2 added
(p.f.u./lOOml)
2320
2116
1885
910
817
160
125
345
87
217
80
77
A1C13 added
(p.f.u./lOO ml)
2260a
1964
1595
990
821a
145
185
377
63a
336
76
69
a Final A1C13 concentration was 0.05 M.
b Data obtained from Tables 3 and 4.
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DISCUSSION
Since the solids of domestic sludges are in significant part fecal material, it seemed
that disruption of the solids by blending might increase the numbers of indigenous virions
exposed to subsequent elution with beef extract. This was not the case. It is possible that
the shear forces exerted by the blender were not strong enough to expose significant
numbers of surfaces to the beef extract eluant. It is also possible that shearing exposed
new receptors that bonded virions previously free in suspension so strongly that they
were not eluted by the beef extract or, alternatively, that the process of blending in-
activated some virions. The small reduction in the numbers of virions recovered by elution
from blended sludges offers some support for the latter possibilities.
Many of the virions indigenous to a sludge are adsorbed on and within the solids of
the sludge, probably on and within the fecal matter with which the virions enter the
sewage. The liquid portion of sludges also contains some virions; therefore, the approach
we chose to use to recover the virions in sludges was one of binding to the sludge solids
those virions free in suspension which allowed us then to deal only with the solids, which
constitute a small percentage of the sludge. Thus, we added divalent (Mg2*) or trivalent
(Al3 +) cations to the sludges to bridge the virions to sludge particles and to neutralize
the negative charges on the virions and on the sludge solids particles so that electro-
static forces could help further to bond the virions to the sludge solids. Moreover, the pH
of the sludges was reduced to 3.0—3.5, below the isoelectric point of many viruses, and
this probably neutralized negative charges on the virions and thereby promoted adsorp-
tion of the virions to the sludge solids (Mix, 1974). The addition of divalent cations
(Mg2 + as MgCl2, final molarity = 0.05) to primary sludges that were subsequently acidi-
fied to pH 3.0—3.5 appeared to increase the numbers of indigenous virions subsequently
eluted beyond those obtained from sludges that were only acidified. The same seemed to
be the case with activated sludges. Moreover, acidification to pH 3.0—3.5 of anaerobic
mesophilically digested sludges to which either divalent (Mg2 + as MgCl2, final molarity
= 0.05) or trivalent (Al3t as A1C13, final molarity = 0.0005) cations had been added al-
most always increased the number of virions eluted subsequently. Thus, either MgCl2 or
A1C13, added in appropriate quantities to sludges subsequently acidified to pH 3.0—3.5,
generally raised the yields of virions subsequently eluted above those obtained in the ab-
sence of either divalent or trivalent cations or in the absence of acidification. Few virions
were ever recovered from the supernatants of centrifuged, acidified, salted sludges. With
buffered 10% BE we have also recovered virions from salted, acidified, anaerobic thermo-
philically digested sludges. Acidification of sludges to pH 3.0-3.5 destroyed about 99% of
the bacteria present and thereby reduced the risk of later contaminating cell cultures used
to detect and quantify the virions recovered.
The volumes of buffered BE used to elute virions from centrifuged sludge solids were
equal to the original volumes of the sludge samples tested. Thus, the procedures des-
cribed are recovery methods and not concentration methods. Large volumes of buf-
fered BE were used for elutions, primarily because extracts from concentrated sludge
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solids were often toxic to cell cultures. To reduce the numbers of cell cultures required
for assays, buffered BE eluates can be reconcentrated by the Katzenelson procedure
(Katzenelson et a]., 197b). Moreover, preliminary data indicate that treatment of cen-
trifuged solids with Freon removes much of the toxicity and may allow elutions to be
undertaken with lesser quantities of buffered BE than we have used thus far. Data demon-
strating the effectiveness of the reconcentration and toxicity reduction procedures will be
published at a later date.
The method described herein, that of fixing suspended virions in a sludge to the sludge
solids by introducing divalent (Mg2+) or trivalent (A13+) cations into the sludge and
acidifying the sludge to pH 3.0-3.5, and eluting the virions from the centrifuged solids
with buffered 10% BE, was designed to recover enteroviruses but will probably recover at
least also the rotaviruses (Farrah et al., 1978). The cell culture or animal host system in
which the virions are detected is, of course, a major factor that determines which virions
will be recovered. In our laboratory, the method we described in this paper was superior
to the beef extract elution procedures that we and others described earlier (Wolf et al.,
1974; Glass et al., 1978; Nielsen and Lydholm, 1980) and better than other procedures
that have been described also (Lund and Hedstrom, 1966; Malina et al., 1975; Turk et
al., 1980; Wellings et al., 1976). A comparative study will be published elsewhere.
ACKNOWLEDGEMENT
Suspended solids were determined for us by the Waste Identification and Analysis
Section, Municipal Environmental Research Laboratory, USEPA, Cincinnati.
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